As described above, our initial attempts to develop an immunotherapeutic regimen for pediatric sarcomas have targeted the tumor specific fusion proteins generated by the t(11,22) and t(2,13) in Ewings sarcoma and alveolar rhabdomyosarcoma respectively. While these targets are attractive due to their potential non-mutability (e.g. they appear to be required for induction of the malignant phenotype) as well as their ultimate tumor specificity, there are several reasons to believe that they may not prove efficacious as a tumor antigen. Firstly, because the unique region is relatively small and limited to the breakpoint region, the likelihood that these epitopes will bind to the wide array of HLA antigens expressed in patients with Ewings sarcoma and alveolar rhabdomyosarcoma is not high. Indeed, in our initial series of patients immunized with immature dendritic cells which have been peptide pulsed with peptides derived from the breakpoint region, some patients have shown no evidence of immune responses despite repeated immunizations. While multiple factors may contribute to this result, it is plausible that deficient binding to HLA alleles could also play a role. Indeed, despite several attempts, no specific binding with these peptides can be demonstrated for the most common HLA alleles. Further, even if binding occurs in some patients, it is unlikely that one particular epitope will prove to function as an immunodominant epitope on the tumor itself across a wide array of HLA alleles. Because of these caveats, we are very interested in identifying new targets and new approaches to immunize patients toward pediatric sarcomas. To this end, we believe that studies in the human system are mandatory, since murine peptide presentation may differ substantially from that observed with similar epitopes in humans. Therefore, current approaches involve attempts to generate primary ex vivo responses to autologous tumor in patients with sarcomas. The experimental set up involves the utilization of autologous monocyte derived dendritic cells, autologous T cells and autologous tumor. We are generating immature dendritic cells using monocytes co-cultured with GM-CSF and IL-4 for three days. At this time, the immature dendritic cells show some upregulation of co- stimulatory molecules, they retain the mannose receptor indicative of phagocytic capacity but are CD83-. Co-culture with apoptotic autologous tumor results in ingestion/phagocytosis of the apoptotic tumor body which can be shown by FACS analysis. Subsequently, the dendritic cells are fully matured using recombinant hCD40 ligand supplies by Immunex, Inc. via CRADA. These fully mature dendritic cells are expected to vigorously express antigens derived from the ingested apoptotic tumor. Co-culture with autologous T cells will be undertaken and attempts to generate anti-tumor effectors will ensue. We will monitor for cytokine production and cytolysis of tumors vs. autologous EBV transformed lymphoblasts or fibroblasts when available. If evidence of cytolytic or cytokine responses to autologous tumor can be shown, subtractive cloning will be used to identify the molecule or molecules which are immunodominant in these responses. In addition, we will search for evidence of translocation specific responses by evaluating for responses to breakpoint peptide pulsed targets and fusion protein transfected EBV lymphoblasts. - dendritic cells, cancer antigens, cancer immunotherapy, Ewing's sarcoma family of tumors (ESFT), rhabdomyosarcoma, - Human Tissues, Fluids, Cells, etc.